Hyperthermus butylicus Medium 865 Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pres- sure–121°C. Solution F: Composition per 10.0mL: Sodium dihydroxybenzoate 0.4g Preparation of Solution F: Add sodium dihydroxybenzoate to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Sparge with 80% N 2 + 20% CO 2 . Solution G: Composition per 10.0mL: Na 2 S·9H 2 O 0.36g Preparation of Solution G: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 . Aseptically and anaerobically combine 920.0mL of sterile solution A, 50.0mL of sterile solution B, 1.0mL of sterile solution C, 1.0mL of sterile solution D, 10.0mL of sterile solution E, 10.0mL of sterile solution F, and 10.0mL of sterile solution G. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Clostridium species. Hydroxybenzoic Acid Medium Composition per liter: Agar 15.0g K 2 HPO 4 ·3H 2 O 4.25g NH 4 Cl 2.0g 4-Hydroxybenzoic acid 1.0g NaH 2 PO 4 ·H 2 O 1.0g MgSO 4 ·7H 2 O 0.2g Nitrilotriacetic acid 0.1g FeSO 4 ·7H 2 O 0.012g MnSO 4 ·H 2 O 3.0mg ZnSO 4 ·7H 2 O 3.0mg CoSO 4 1.0mg pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add 4-hydroxybenzoic acid and nitrilo- triacetic acid to approximately 600.0mL of distilled/deionized water. Adjust pH to 8.0 with concentrated NaOH. Add remaining compo- nents. Mix thoroughly. Readjust pH to 7.2. Bring volume to 1.0L with distilled/deionized water. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Bacillus species. Hydroxybutyrate Medium (3HB Medium) (LMG Medium 186) Composition per liter: Agar 20.0g DL-3-hydroxybutyrate 3.0g NH 4 Cl 1.0g MgSO 4 ·7H 2 O 0.5g Ferric ammonium citrate 50.0mg Yeast extract 50.0mg Buffer solution 333.3mL pH 6.8 ± 0.2 at 25°C Buffer Solution: KH 2 PO 4 0.45g Na 2 HPO 4 ·12 H 2 O 2.39g Preparation of Buffer Solution: Add components to distilled/de- ionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Paucimonas lemoignei. Hyperthermus butylicus Medium Composition per 1010.0mL: NaCl 17.0g Pancreatic digest of casein 6.0g Sulfur, powdered 6.0g MgSO 4 ·7H 2 O 3.5g MgCl 2 ·6H 2 O 2.75g NiCl 2 ·6H 2 O 2.0g Yeast extract 2.0g CaCl 2 ·2H 2 O 0.75g KH 2 PO 4 0.5g NH 4 Cl 0.5g KCl 0.325g NaBr 0.05g H 3 BO 3 0.015g (NH 4 ) 2 SO 4 10.0mg SrCl 2 ·6H 2 O 7.5mg Citric acid 5.0mg KI 2.5mg Resazurin 1.0mg Trace elements solution 10.0mL Na 2 S·9H 2 O solution 10.0mL pH 6.0–6.5 at 25°C Trace Elements Solution: Composition per liter: MgSO 4 ·7 H 2 O 3.0g Nitrilotriacetic acid 1.5 g CaCl 2 ·2 H 2 O .1.0g NaCl 1.0g MnSO 4 ·2 H 2 O 0.5g CoSO 4 ·7 H 2 O 0.18g ZnSO 4 ·7 H 2 O 0.18g FeSO 4 ·7 H 2 O 0.1g NiCl 2 ·6 H 2 O 0.025g KAI(SO 4 ) 2 ·12 H 2 O 0.02g CuSO 4 ·5 H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2 H 2 O 0.01g Na 2 SeO 3 ·5 H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Adjust pH to 7.0 with KOH. Add distilled/deionized water to 1.0L. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.5g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. © 2010 by Taylor and Francis Group, LLC 866 Hyphomicrobium Enrichment Medium Gas under 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pres- sure–121°C. Preparation of Medium: Add components, except Na 2 S·9H 2 O solu- tion, to distilled/deionized water and bring volume to 1.0L. Mix thorough- ly. Adjust pH to 6.0–6.5 with 6N H 2 SO 4 . Sparge wih 100% N 2 . Sterilize by bringing to 90°C for 60 min on 3 consecutive days. Immediately prior to inoculation, add 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thor- oughly. Use: For the cultivation and maintenance of Hyperthermus butylicus. Hyphomicrobium Enrichment Medium Composition per 100.0mL: KNO 3 0.04g Na 2 HPO 4 ·7H 2 O 0.02g MgSO 4 ·7H 2 O 0.48mg FeCl 3 ·7H 2 O 0.02mg MnCl 2 ·4H 2 O 0.01mg pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized wa- ter and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and enrichment of Hyphomicrobium species. Hyphomicrobium Medium Composition per liter: Agar 15.0g Na 2 HPO 4 2.13g KH 2 PO 4 1.36g MgSO 4 ·7H 2 O 0.2g CaCl 2 ·2H 2 O 9.95mg FeSO 4 ·7H 2 O 5.0mg MnSO 4 ·4H 2 O 2.5mg Na 2 MoO 4 ·2H 2 O 2.5mg Urea solution 30.0mL Methanol 4.0mL Urea Solution: Composition per 100.0mL: Urea 20.0g Preparation of Urea Solution: Add urea to distilled/deionized wa- ter and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Filter sterilize methanol. Add compo- nents, except urea solution and methanol, to distilled/deionized water and bring volume to 966.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile urea solution and sterile methanol. Mix thoroughly. Aseptically distribute into sterile tubes or bottles. Use: For the cultivation of Hyphomicrobium species. Hyphomicrobium Medium Composition per liter: Noble agar 18.0g Na 2 HPO 4 2.15g KH 2 PO 4 1.36g (NH 4 ) 2 SO 4 0.5g MgSO 4 ·7H 2 O 0.2g Trace elements solution 5.0mL Methylamine·HCl solution 20.0mL pH 7.1 ± 0.1 at 25°C Trace Elements Solution: Composition per 100.0mL: CuCl 2 0.15g FeSO 4 ·7H 2 O 0.1g Na 2 MoO 4 ·2H 2 O 0.05g MnSO 4 ·H 2 O 0.035g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thorough- ly. Methylamine·HCl Solution: Composition per 20.0mL: Methylamine·HCl 3.38g Preparation of Methylamine·HCl Solution: Add methyl- amine·HCl to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except methylamine·HCl solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile methylamine·HCl solution. Mix thoroughly. Adjust pH to 7.1, if necessary. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Hyphomicrobium aestu- arii, Hyphomicrobium facilis, Hyphomicrobium hollandicum, Hypho- microbium vulgare, and Hyphomicrobium zavarzinii. Hyphomicrobium Medium 337a Composition per liter: KH 2 PO 4 1.3g Na 2 HPO 4 1.13g (NH 4 ) 2 SO 4 0.5g MgSO 4 ·7H 2 O 0.2g CaCl 2 ·2H 2 O 3.09mg FeSO 4 ·7H 2 O 2.0mg Na 2 MoO 4 ·2H 2 O 1.0mg MnSO 4 ·4H 2 O 0.88mg pH 7.2–7.5 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the enrichment and cultivation of Hyphomicrobium species. Hyphomicrobium methylovorum Medium Composition per liter: Agar 15.0g (NH 4 ) 2 HPO 4 3.0g NaCl 1.0g MgSO 4 ·7H 2 O 0.2g FeSO 4 ·7H 2 O 10.0mg MnSO 4 ·2H 2 O 5.0mg Tap water 1.0L Methanol 10.0mL Vitamin mixture 5.0mL Vitamin Mixture: Composition per liter: Inositol 200.0mg Choline 100.0mg Calcium DL-pantothenate 40.0mg © 2010 by Taylor and Francis Group, LLC Hypoxanthine Agar 867 Niacin 40.0mg Pyridoxine·HCl 40.0mg Riboflavin 40.0mg p-Aminobenzoic acid 20.0mg Thiamine·HCl 20.0mg Biotin 0.2mg Folic acid 0.2mg Cyanocobalamin 2.0μg Preparation of Vitamin Mixture: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter ster- ilize. Preparation of Methanol: Filter sterilize 10.0mL of methanol. Preparation of Medium: Add components, except methanol and vi- tamin mixture, to distilled/deionized water and bring volume to 985.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Asepti- cally add 10.0mL of sterile methanol and 5.0mL of sterile vitamin mixture. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Hyphomicrobium methylovorum. Hyphomicrobium Strain X Agar Composition per liter: Agar 15.0g Methylamine·HCl 3.4g K 2 HPO 4 1.55g (NH 4 ) 2 SO 4 1.0g NaH 2 PO 4 ·H 2 O 0.5g MgSO 4 ·7H 2 O 0.2g Trace elements solution 0.2mL pH 7.2 ± 0.2 at 25°C Trace Elements Solution: Composition per liter: Disodium EDTA 50.0g ZnSO 4 ·7H 2 O 22.0g CaCl 2 ·2H 2 O 5.54g MnCl 2 ·4H 2 O 5.06g FeSO 4 ·7H 2 O 5.0g CoCl 2 ·6H 2 O 1.61g CuSO 4 ·5H 2 O 1.57g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Adjust pH to 7.0 with KOH. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Distribute into tubes or flasks. Auto- clave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Hyphomicrobium species. Hyphomicrobium Strain X Broth Composition per liter: K 2 HPO 4 1.55g (NH 4 ) 2 SO 4 1.0g Methylamine·HCl 0.7g NaH 2 PO 4 ·H 2 O 0.5g MgSO 4 ·7H 2 O 0.2g Trace elements solution 0.2mL pH 7.2 ± 0.2 at 25°C Trace Elements Solution: Composition per liter: Disodium EDTA 50.0g ZnSO 4 ·7H 2 O 22.0g CaCl 2 ·2H 2 O 5.54g MnCl 2 ·4H 2 O 5.06g FeSO 4 ·7H 2 O 5.0g CoCl 2 ·6H 2 O 1.61g CuSO 4 ·5H 2 O 1.57g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Adjust pH to 7.0 with KOH. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Distribute into tubes or flasks. Auto- clave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Hyphomicrobium species. Hyphomonas Enrichment Medium Composition per liter: Peptone 0.05g Yeast extract 0.05g Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the isolation and cultivation of Hyphomonas species. Hyphomonas Medium Composition per liter: Pancreatic digest of casein 2.0g MgCl 2 ·2H 2 O 2.0g Yeast extract 1.0g pH 7.5 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5 us- ing indicator paper. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenence of Hyphomonas polymorpha. Hypoxanthine Agar Composition per 1100.0mL: Agar 15.0g Peptone 5.0g Hypoxanthine solution 5.0g Beef extract 3.0g pH 7.0 ± 0.1 at 25°C Hypoxanthine Solution: Composition per 100.0mL: Hypoxanthine 5.0g Preparation of Hypoxanthine Solution: Add hypoxanthine to distilled/deionized water and bring volume to 100.0mL. Mix thorough- ly. Filter sterilize. Preparation of Medium: Add components, except hypoxanthine solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of sterile hypoxanthine solu- © 2010 by Taylor and Francis Group, LLC 868 Idiomarina Medium tion. Mix thoroughly. Pour into sterile 15mm × 100mm Petri dishes in 25.0mL volumes. Use: For the cultivation and differentiation of bacteria based on hypoxanthine hydrolysis. Bacteria that hydrolyze hypoxanthine, such as Streptomyces griseus, appear with a clear zone under and around the colonies. Nocardia asteroides does not hydrolyze hypoxanthine. IBB Agar See: Inositol Brilliant Green Bile Salts Agar Idiomarina Medium (DSMZ Medium 1016) Composition per liter: NaCl 30.0-60.0g Glucose 10.0g Proteose peptone 5.0g Yeast extract 3.0g Malt extract 3.0g pH 7.0 ± 0.2 at 25°C Preparation of Medium: Add componentsto distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Idiomarina spp. IE Medium Composition per 1011.0mL: Agar 15.0g Peptone 5.0g Yeast extract 1.0g Basal salts solution 1.0L Lactose solution 10.0g Trace elements solution 1.0mL Basal Salts Solution: Composition per liter: MgSO 4 0.5g Phosphate solution 20.0mL (NH 4 )SO 4 (36% solution) 5.0mL Phosphate Solution: Composition per liter: K 2 HPO 4 95.0g NaH 2 PO 4 ·2H 2 O 78.0g Preparation of Phosphate Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Basal Salts Medium: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 6.8-7.0. Lactose Solution: Composition per 10.0mL: Lactose 2.5g Preparation of Lactose Solution: Add lactose to distilled/deion- ized water and bring volume to 10.0mL. Mix thoroughly. Filter steril- ize. Warm to 50°–55°C. Trace Elements Solution: Composition per liter: Disodium EDTA 0.5g FeSO 4 ·7H 2 O 0.2g H 3 BO 3 0.03g CoCl 2 ·6H 2 O 0.02g ZnSO 4 ·7H 2 O 0.01g MnCl 2 ·4H 2 O 3.0mg Na 2 MoO 4 ·2H 2 O 3.0mg NiCl 2 ·6H 2 O 2.0mg CaCl 2 ·2H 2 O 1.0mg Preparation of Trace Elements Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Au- toclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Combine components, except lactose so- lution and trace elements solution. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 10.0mL of sterile lactose solution and 1.0mL of sterile trace elements solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Bacillus species. IFO Agar Composition per liter: Agar 20.0g (NH 4 ) 2 HPO 4 3.0g NaCl 1.0g MgSO 4 ·7H 2 O 0.2g FeSO 4 ·7H 2 O 10.0mg MnSO 4 ·6H 2 O 5.0mg Riboflavin 0.02mg Calcium pantothenate 0.02mg Pyridoxine·HCl 0.02mg Nicotinic acid 0.02mg p-Aminobenzoic acid 0.01mg Thiamine·HCl 0.01mg Biotin 1.0μg Methanol 10.0mL pH 7.0 ± 0.2 at 25°C Preparation of Medium: Add components, except agar and meth- anol, to distilled/deionized water and bring volume to 490.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. In a separate flask, add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically combine the two sterile solutions. Aseptically add 10.0mL of filter-sterilized methanol. Mix thoroughly. Adjust pH to 7.0. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Hyphomicrobium methy- lovorum. IFO Broth Composition per liter: (NH 4 ) 2 HPO 4 3.0g NaCl 1.0g MgSO 4 ·7H 2 O 0.2g FeSO 4 ·7H 2 O 10.0mg MnSO 4 ·6H 2 O 5.0mg Riboflavin 20.0μg Calcium pantothenate 20.0μg Pyridoxine·HCl 20.0μg Nicotinic acid 20.0μg © 2010 by Taylor and Francis Group, LLC Ignisphaera Medium 869 p-Aminobenzoic acid 10.0μg Thiamine·HCl 10.0μg Biotin 1.0μg Methanol 10.0mL pH 7.0 ± 0.2 at 25°C Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of filter-sterilized methanol. Mix thoroughly. Adjust pH to 7.0. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Hyphomicrobium methy- lovorum. IFO Medium 802 Composition per liter: Polypeptone™ 10.0g Yeast extract 2.0g MgSO 4 ·7H 2 O 1.0g pH 7.0 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Dis- tribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Sphingomonas asaccharolytica, Sphin- gomonas pruni, Sphingomonas mali, and Sphingomonas rosa. Ignicoccus Medium (DSMZ Medium 897) Composition per liter: NaCl 13.65g Sulfur, powdered 5.0g MgSO 4 ·7H 2 O 3.5g MgCl 2 ·6H 2 O 2.75g Meat extract 1.0g KH 2 PO 4 0.5g CaCl 2 ·2H 2 O 0.38g KCl 0.33g (NH 4 ) 2 SO 4 0.25g NaBr 0.05g H 3 BO 3 15.0mg SrCl 3 ·6H 2 O 7.50mg KI 0.05mg Resazurin 0.5mg Na 2 S·9H 2 O solution 10.0mL pH 5.5 ± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.2g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Sparge with N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an- aerobically. Preparation of Medium: Add components, except Na 2 S·9H 2 O so- lution, to distilled/deionized water and bring volume to 990.0mL. Sparge medium with N 2 gas for 30–60 min. Mix thoroughly. Add 10.0mL Na 2 S·9H 2 O solution. Mix thoroughly. Adjust pH to 5.5 with H 2 SO 4 . Distribute into tubes or bottles under 80% H 2 and 20% CO 2 gas mixture. Heat the vessels containing medium in boiling water for 1 hr before inoculation. After inoculation pressurize the vessels with 80% H 2 and 20% CO 2 gas mixture to 2 bar overpressure. Use: For the cultivation of Ignicoccus islandicus and Ignicoccus pacifi- cus. Ignisphaera Medium (DSMZ Medium 1043) Composition per liter: Trypticase peptone 2.0g Starch, soluble 2.0g (NH 4 ) 2 SO 4 1.3g MgSO 4 ·7H 2 O 0.28g KH 2 PO 4 0.28g Yeast extract 0.1g L-Cysteine 0.3g CaCl 2 ·2H 2 O 74.0mg Resazurin 0.5mg FeCl 2 ·6H 2 O 0.5mg Trace elements solution 10.0mL FeCl 2 solution 10.0mL Na 2 S·9H 2 O solution 10.0mL pH 6.5 ± 0.2 at 25°C FeCl 2 Solution: Composition per 10.0mL: FeCl 2 ·6H 2 O 0.5mg Preparation of FeCl 2 Solution: Add FeCl 2 ·6H 2 O to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g H 3 BO 3 0.01g Na 2 MoO 4 ·4H 2 O 0.01g CuSO 4 ·5H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Preparation of Medium: Add components, except iron chloride and sulfide solutions, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 1 © 2010 by Taylor and Francis Group, LLC 870 Ilyobacter Broth min. Cool to room temperature while sparging with 100% N 2 . Add FeCl 3 solution. Adjust pH to 6.3. Dispense under an atmosphere of 100% N 2 into suitable culture vessels (e.g., aliquots of 20mL medium into 50mL serum bottles). Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Adjust pH to 6.5. Prior to inocula- tion aseptically and anoxically add sulfide solution. Use: For the cultivation of Ignisphaera spp. IGP Medium See: Intracellular Growth Phase Medium Ilyobacter Agar Composition per liter: NaCl 20.0g Agar 15.0g MgCl 2 ·6H 2 O 3.0g KCl 0.5g NH 4 Cl 0.25g KH 2 PO 4 0.2g CaCl 2 ·2H 2 O 0.15g Resazurin 1.0mg Sodium sulfide solution 10.0mL Sodium L-tartrate solution 10.0mL NaHCO 3 solution 10.0mL Trace elements solution SL-7 1.0mL pH 7.2 ± 0.2 at 25°C Sodium Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 3.6g Preparation of Sodium Sulfide Solution: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C under N 2 . Maintain under 100% N 2 . Sodium L-Tartrate Solution: Composition per 10.0mL: Sodium L-tartrate 2.0g Preparation of Sodium L-Tartrate Solution: Add sodium L-tar- trate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Maintain under 80% N 2 + 20% CO 2 . NaHCO 3 Solution: Composition per 10.0mL: NaHCO 3 2.5g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Filter ster- ilize. Maintain under 80% N 2 + 20% CO 2 . Trace Elements Solution SL-7: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·4H 2 O 0.1g ZnCl 2 0.07g H 3 BO 3 0.062g Na 2 MoO 4 ·2H 2 O 0.036g NiCl 2 ·6H 2 O 0.024g CuCl 2 ·2H 2 O 0.017g HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-7: Add the FeCl 2 ·4H 2 O to the HCl. Add distilled/deionized water and bring vol- ume to 1.0L. Add remaining components. Mix thoroughly. Filter ster- ilize. Maintain under 80% N 2 + 20% CO 2 . Preparation of Medium: Add components—except agar, sodium sulfide solution, sodium L-tartrate solution, NaHCO 3 solution, and trace elements solution SL-7—to distilled/deionized water and bring volume to 469.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Maintain under 80% N 2 + 20% CO 2 . Aseptically add 10.0mL of sodi- um L-tartrate solution, 10.0mL of NaHCO 3 solution, and 1.0mL of trace elements solution SL-7 under 80% N 2 + 20% CO 2 . Mix thorough- ly. In a separate flask, add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Combine sterile agar and sterile basal medium. Adjust pH to 7.2. Asep- tically add 10.0mL of sodium sulfide solution. Pour into sterile Petri dishes or distribute into sterile tubes. Maintain under 80% N 2 + 20% CO 2 . Use: For the cultivation and maintenance of Ilyobacter tartaricus. Ilyobacter Broth Composition per liter: NaCl 20.0g MgCl 2 ·6H 2 O 3.0g KCl 0.5g NH 4 Cl 0.25g KH 2 PO 4 0.2g CaCl 2 ·2H 2 O 0.15g Resazurin 1.0mg Sodium sulfide solution 10.0mL Sodium L-tartrate solution 10.0mL NaHCO 3 solution 10.0mL Trace elements solution SL-7 1.0mL pH 7.2 ± 0.2 at 25°C Sodium Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 3.6g Preparation of Sodium Sulfide Solution: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C under 100% N 2 . Maintain under 100% N 2 . Sodium L-Tartrate Solution: Composition per 10.0mL: Sodium L-tartrate 2.0g Preparation of Sodium L-Tartrate Solution: Add sodium L-tar- trate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Maintain under 80% N 2 + 20% CO 2 . NaHCO 3 Solution: Composition per 10.0mL: NaHCO 3 2.5g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Filter ster- ilize. Maintain under 80% N 2 + 20% CO 2 . Trace Elements Solution SL-7: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·4H 2 O 0.1g ZnCl 2 0.07g © 2010 by Taylor and Francis Group, LLC Ilyobacter polytropus Medium 871 H 3 BO 3 0.062g Na 2 MoO 4 ·2H 2 O 0.036g NiCl 2 ·6H 2 O 0.024g CuCl 2 ·2H 2 O 0.017g HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-7: Add the FeCl 2 ·4H 2 O to the HCl. Add distilled/deionized water and bring vol- ume to 1.0L. Add remaining components. Mix thoroughly. Filter ster- ilize. Maintain under 80% N 2 + 20% CO 2 . Preparation of Medium: Add components—except sodium sulfide solution, sodium L-tartrate solution, NaHCO 3 solution, and trace ele- ments solution SL-7—to distilled/deionized water and bring volume to 969.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Maintain un- der 80% N 2 + 20% CO 2 . Aseptically add 10.0mL of sodium L-tartrate solution, 10.0mL of NaHCO 3 solution, and 1.0mL of trace elements solution SL-7 under 80% N 2 + 20% CO 2 . Mix thoroughly. Aseptically distribute into sterile tubes or flasks under 80% N 2 + 20% CO 2 . Adjust pH to 7.2. At time of inoculation add sodium sulfide solution to a final concentration of 0.1%. Use: For the cultivation and maintenance of Ilyobacter tartaricus. Ilyobacter Medium Composition per liter: Crotonic acid 1.7g NaCl 1.0g Yeast extract 1.0g Na 2 HPO 4 ·12H 2 O 0.7g KCl 0.5g MgCl 2 ·6H 2 O 0.4g NH 4 Cl 0.3g Na 2 SO 4 0.1g Sodium sulfide solution 10.0mL CaCl 2 ·2H 2 O (1.0% ) 1.0mL FeCl 3 (0.5% ) 1.0mL Modified SL-7 trace elements solution 1.0mL Resazurin (0.1% ) 1.0mL Selenite-tungstate solution 1.0mL pH 6.8–7.2 at 25°C Sodium Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 3.6g Preparation of Sodium Sulfide Solution: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C under N 2 . Maintain under 100% N 2 . Modified SL-7 Trace Elements Solution: Composition per liter: CoCl 2 ·6H 2 O 0.2g MnCl 2 ·4H 2 O 0.1g ZnCl 2 0.07g H 3 BO 3 0.06g Na 2 MoO 4 ·2H 2 O 0.04g CuCl 2 ·2H 2 O 0.02g NiCl 2 ·6H 2 O 0.02g HCl (1N) 3.0mL Preparation of Modified SL-7 Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Maintain under 80% N 2 + 20% CO 2 . Selenite-Tungstate Solution: Composition per liter: NaOH 0.5g Na 2 WO 4 ·2H 2 O 4.0mg Na 2 SeO 3 ·5HO 3.0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Maintain under 80% N 2 + 20% CO 2 . Preparation of Medium: Add components—except sodium sulfide solution, modified SL-7 trace elements solution, and selenite-tungstate solution—to distilled/deionized water and bring volume to 969.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Adjust pH to 5.5. Cool to 45°–50°C under 100% N 2 . Maintain under 100% N 2 . Aseptically add 1.0mL of sterile modified SL-7 trace elements solution and 1.0mL of sterile selenite- tungstate solution under 100% N 2 . Mix thoroughly. Aseptically distrib- ute into sterile tubes or flasks under 100% N 2 . At time of inoculation, add sodium sulfide solution to a final concentration of 1.0%. Maintain under 100% N 2 . Use: For the cultivation and maintenance of Ilyobacter delafieldii. Ilyobacter polytropus Medium Composition per 1011.0mL: Solution A 1.0L Solution B 10.0mL Vitamin solution 1.0mL pH 7.2–7.4 at 25°C Solution A: Composition per liter: NaHCO 3 4.5g Na 2 SO 4 2.84g Sodium 3-hydroxybutyrate 1.3g NaCl 1.17g Yeast extract 1.0g MgCl 2 ·6H 2 O 0.4g KCl 0.3g NH 4 Cl 0.27g KH 2 PO 4 0.2g CaCl 2 ·2H 2 O 0.15g Resazurin 0.5mg Trace elements solution 1.0mL Preparation of Solution A: Add components, except NaHCO 3 and vitamin solution, and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 to 4 min. Allow to cool to room temperature while gassing under O 2 -free 80% N 2 + 20% CO 2 . Add NaHCO 3 and continue gassing with O 2 -free 80% N 2 + 20% CO 2 until pH reaches 6.9–7.1. Seal the flask under 80% N 2 + 20% CO 2 . Au- toclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 120.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 68.0mg H 3 BO 3 62.0mg Na 2 MoO 4 ·2H 2 O 24.0mg NiCl 2 ·6H 2 O 24.0mg CuCl 2 ·2H 2 O 17.0mg HCl (25% solution) 10.0mL © 2010 by Taylor and Francis Group, LLC 872 Ilyobacter tartaricus Medium Preparation of Trace Elements Solution: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized wa- ter and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Gas under 100% N 2 . Vitamin Solution: Composition per 100.0mL: Thiamine·HCl 10.0mg p-Aminobenzoic acid 4.0mg D(+)-Biotin 1.0mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Gas under 100% N 2 . Solution B: Composition per 10.0mL: Na 2 S·9H 2 O 0.36g Preparation of Solution B: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Gas under 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: To 1.0L of sterile solution A, add 10.0mL of sterile solution B and 1.0mL of sterile vitamin solution. Mix thor- oughly. Adjust final pH to 7.2–7.4. Use: For the cultivation and maintenance of Ilyobacter polytropus. Ilyobacter tartaricus Medium Composition per liter: NaCl 20.0g MgCl 2 ·6H 2 O 3.0g KCl 0.5g NH 4 Cl 0.25g KH 2 PO 4 0.2g CaCl 2 ·2H 2 O 0.15g Resazurin 1.0mg NaHCO 3 solution 20.0mL Sodium L-tartrate solution 10.0mL Na 2 S·9H 2 O solution 10.0mL Trace elements solution SL-10 1.0mL pH 7.2 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 20.0mL: NaHCO 3 2.5g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 20.0mL. Mix thoroughly. Filter ster- ilize. Gas under 80% N 2 + 20% CO 2 . Sodium L-Tartrate Solution: Composition per 10.0mL: Sodium L-tartrate 2.0g Preparation of Sodium L-Tartrate Solution: Add sodium L-tar- trate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Gas under 80% N 2 + 20% CO 2 . Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.36g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Gas under 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Gas under 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Preparation of Medium: Add components, except NaHCO 3 solu- tion, sodium L-tartrate solution, and Na 2 S·9H 2 O solution, to distilled/de- ionized water and bring volume to 960.0mL. Mix thoroughly. Gas under 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 20.0mL of sterile NaHCO 3 solution, 10.0mL of sterile sodium L-tartrate solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Ilyobacter tartaricus. Ilyobacter tartaricus Medium Composition per liter: NaCl 20.0g MgCl 2 ·6H 2 O 3.0g KCl 0.5g NH 4 Cl 0.25g KH 2 PO 4 0.2g CaCl 2 ·2H 2 O 0.15g Resazurin 1.0mg NaHCO 3 solution 20.0mL Sodium L-tartrate solution 10.0mL Na 2 S·9H 2 O solution 10.0mL Yeast extract solution 10.0mL Trace elements solution SL-10 1.0mL pH 7.2 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 20.0mL: NaHCO 3 2.5g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 20.0mL. Mix thoroughly. Filter ster- ilize. Gas under 80% N 2 + 20% CO 2 . Sodium L-Tartrate Solution: Composition per 10.0mL: Sodium L-tartrate 2.0g Preparation of Sodium L-Tartrate Solution: Add sodium L-tar- trate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Gas under 80% N 2 + 20% CO 2 . Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.36g © 2010 by Taylor and Francis Group, LLC Imidazole Utilization Medium 873 Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Gas under 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Yeast Extract Solution: Composition per 10.0mL: Yeast extract 1.0g Preparation of Yeast Extract Solution: Add yeast extract to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Gas under 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Gas under 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Preparation of Medium: Add components, except NaHCO 3 solu- tion, sodium L-tartrate solution, yeast extract solution, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gas under 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 20.0mL of sterile NaHCO 3 solution, 10.0mL of sterile sodium L-tartrate solution, 10.0mL of sterile yeast extract solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Propionigenium modes- tum. IM See: Infection Medium Imhoff’s Medium, Modified Composition per liter: NaCl 30.0g NaHCO 3 3.0g KH 2 PO 4 1.0g NH 4 Cl 1.0g Sodium acetate 1.0g Na 2 SO 4 0.7g MgCl 2 ·6H 2 O 0.5g Sodium ascorbate 0.5g CaCl 2 ·2H 2 O 0.1g Yeast extract 0.1g Sodium sulfide solution 10.0mL SLA trace elements solution 1.0mL VA vitamin solution 1.0mL pH 6.9–7.0 at 25°C Sodium Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 2.0g Preparation of Sodium Sulfide Solution: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C under N 2 . Maintain under 100% N 2 . SLA Trace Elements Solution: Composition per liter: FeCl 2 ·4H 2 O 1.8g H 3 BO 3 0.5g CoCl 2 ·6H 2 O 0.25g ZnCl 2 0.1g MnCl 2 ·4H 2 O 0.07g Na 2 MoO 4 ·2H 2 O 0.03g CuCl 2 ·2H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.01g NiCl 2 ·6H 2 O 0.01g Preparation of SLA Trace Elements Solution: Add compo- nents to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Adjust pH to 2–3. Filter sterilize. VA Vitamin Solution: Composition per 500.0mL: Nicotinamide 0.17g Thiamine·HCl 0.15g p-Aminobenzoic acid 0.1g Biotin 0.05g Calcium pantothenate 0.05g Pyridoxine·2HCl 0.05g Cyanocobalamin 0.02g Preparation of VA Vitamin Solution: Add components to dis- tilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components—except sodium sulfide solution, SLA trace elements solution, and VA vitamin solution—to distilled/deionized water and bring volume to 988.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Asep- tically add 1.0mL of sterile SLA trace elements solution and 1.0mL of sterile VA vitamin solution. Aseptically add 10.0mL of sterile sodium sulfide solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Rhodobacter adriaticus and Rhodobacter sulfidophilus. Imidazole Utilization Medium Composition per liter: Imidazole 5.0g KH 2 PO 4 0.5g MgSO 4 ·7H 2 O 0.5g CaCl 2 3.0mg FeSO 4 ·7H 2 O 3.0mg Molybdenum solution 1.0mL Trace elements solution 1.0mL pH 6.0 ± 0.2 at 25°C Molybdenum Solution: Composition per 18.0mL: Na 2 MoO 4 ·2H 2 O 0.5mg Preparation of Molybdenum Solution: Add components to dis- tilled/deionized water and bring volume to 18.0mL. Mix thoroughly. Filter sterilize. © 2010 by Taylor and Francis Group, LLC 874 Indole Medium Trace Elements Solution: Composition per 18.0mL: H 3 BO 3 11.0mg MnCl 2 ·4H 2 O 7.0mg Al 2 (SO 4 ) 3 ·18 H 2 O 1.94mg Co(NO 3 ) 2 ·6H 2 O 1.0mg CuSO 4 ·5H 2 O 1.0mg NiSO 4 ·6H 2 O 1.0mg ZnSO 4 ·H 2 O 0.62mg KBr 0.5mg KI 0.5mg LiCl 0.5mg SnCl 2 ·2H 2 O 0.5mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 18.0mL. Mix thorough- ly. Filter sterilize. Preparation of Medium: Add components, except molybdenum solution and trace elements solution, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 1.0mL of molybdenum solution and 1.0mL of trace el- ements solution. Mix thoroughly. Adjust pH to 6.0 with phosphoric ac- id. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Pseudomonas species. Indole Medium Composition per 200.0mL: K 2 HPO 4 3.13g L-Tryptophan 1.0g NaCl 1.0g KH 2 PO 4 0.27g pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 200.0mL. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the differentiation of microorganisms by means of indole production from the tryptophan test. Indole Medium Composition per liter: Pancreatic digest of casein 20.0g pH 7.3 ± 0.2 at 25°C Preparation of Medium: Add pancreatic digest of casein to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Dis- tribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the differentiation of microorganisms by means of the indole test. Indole Medium, CDC (BAM M65) Composition per liter: Pancreatic digest of casein 20.0g pH 7.3 ± 0.2 at 25°C Preparation of Medium: Add pancreatic digest of casein to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Dis- tribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the differentiation of microorganisms by means of the indole test. Indole Nitrate HiVeg Medium (Tryptone Nitrate HiVeg Medium) Composition per liter: Plant hydrolysate 20.0g Na 2 HPO 4 2.0g Agar 1.0g Glucose 1.0g Potassium nitrate 1.0g pH 7.2 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Hi- Media. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling with frequent agitation. Distribute into tubes or flasks. Auto- clave for 15 min at 15 psi pressure–121°C. Use: For the identification of microorganisms by means of the nitrate reduction and indole tests. Indole Nitrate Medium (Trypticase™ Nitrate Broth) Composition per liter: Pancreatic digest of casein 20.0g Na 2 HPO 4 2.0g Agar 1.0g Glucose 1.0g KNO 3 1.0g pH 7.2 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling with frequent agitation. Distribute into tubes or flasks. Auto- clave for 15 min at 15 psi pressure–121°C. Use: For the identification of microorganisms by means of the nitrate reduction and indole tests. Infection Medium (IM) Composition per 100.0mL: Pancreatic digest of gelatin 0.05g Bile salts No. 3 0.05g Brain heart, solids from infusion 0.02g Peptic digest of animal tissue 0.02g NaCl 0.017g Glucose 0.01g Na 2 HPO 4 8.0mg Earle’s balanced salts solution 80.0mL Fetal bovine serum, heat inactivated (2 hr at 55°C) 20.0mL pH 7.4 ± 0.2 at 25°C Earle’s Balanced Salts Solution: Composition per liter: NaCl 6.8g NaHCO 3 2.2g © 2010 by Taylor and Francis Group, LLC . pressure–121°C. Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 . Aseptically and anaerobically combine 920.0mL of sterile solution A, 50.0mL of sterile solution B, 1.0mL of sterile. sterile solution B, 1.0mL of sterile solution C, 1.0mL of sterile solution D, 10.0mL of sterile solution E, 10.0mL of sterile solution F, and 10.0mL of sterile solution G. Mix thoroughly. Aseptically and. 1.0L of sterile solution A, add 10.0mL of sterile solution B and 1.0mL of sterile vitamin solution. Mix thor- oughly. Adjust final pH to 7.2–7.4. Use: For the cultivation and maintenance of Ilyobacter